We have previously shown a time-of-day variation in the response of systolic [Ca2+]i to the non-specific ß-adrenergic (ß-ADR) agonist isoproterenol (ISO), which is linked to a variation in Nitric Oxide (NO) signalling . This may reflect stimulation of ß3-ADRs, which induces a NO-dependent negative inotropic response . As action potential duration (APD) impacts systolic [Ca2+]i and a time-of-day variation exists in the regulation AP , we investigated the effect of ß3-ADR stimulation on the APD and systolic [Ca2+]i.
Ventricular myocytes were isolated by enzymatic digestion of male Wistar rat hearts at two time points; 3 h into the rest-period and active-period. [Ca2+]i was measured in myocytes loaded with Fura-2. APD was measured using whole-cell patch-clamp recording. I to and I LTCC current density were measured using the whole-cell voltage-clamp technique. Data shown as number of hearts (number of cells), mean ± S.E.M., 2-way ANOVA, Tukey’s post hoc test.
In order to determine whether the previously shown time-of-day variation in response to ISO can be explained by a variation in ß3-ADR dependant NOS signalling, we investigated systolic [Ca2+]i in rest and active-period myocytes in response to the specific ß3-ADR agonist BRL37344. BRL37344 (200 nM) resulted in a significant reduction in systolic [Ca2+]i in rest-period myocytes, from 458.5 ± 41.2 nM to 361.2 ± 18.0 nM (n = 3–4 (18–19), P < 0.001) but had no significant effect on active period myocytes.
BRL37344 also significantly reduced APD30, 18.3 ± 2.2 ms to 14.4 ± 1.6 ms (P < 0.001), and APD50, 32.9 ± 4.3 ms to 26.5 ± 3.1 ms, (P < 0.001) in rest-period myocytes (n = 3–4 (12–15) with no significant change in either APD30/50 in active-period myocytes.
I to current density was measured in rest-period myocytes using K+ electrode solution; cell perfusate contained 200 nM CdCl2 to block Ca2+ currents. BRL37344 caused a significant drop in I to density at +30 mV, from 6.38 ± 0.90 to 4.59 ± 0.11 pA/pF (n = 4 (13), P < 0.05). There was a small but non-significant recovery of the reduction due to BRL following NOS inhibition with L-NNA.
I LTCC current density was measured in rest-period myocytes using Cs electrode solution to block K+ currents. Stimulation of ß3-ADR with BRL37344 caused no significant difference in either peak LTCC current density, from -7.46 ± -0.50 to -8.09 ± -0.80 pA/pF, or in the voltage dependence of the peak Ca2+ current, 0mV (n = 3 (13)).
The time-of-day variation we have described previously is seen a decreased [Ca2+]i sensitivity during the active-period. Therefore, our data suggests that ß3-ADR dependant NOS signalling is not likely to be responsible for this time-of-day variation as BRL37344 caused a significant drop in [Ca2+]i and APD30/50 in rest-period myocytes only. Further investigation of the effect of BRL37344 on rest-period myocytes appears to show the reduction in [Ca2+]i and APD30/50 is not due to an increase in the I to or to a reduction in I LTCC.
- sympathetic stimulation
- nitric oxide
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